Image based correction for unwanted light signals in a specific region of interest

ABSTRACT

A method for correcting the signal in an image having a plurality of regions of interest, the method comprising the steps of: (a) providing an image having a plurality of regions of interest, these regions of interest having areas between them; (b) determining a region of correction between at least two regions of interest; (c) calculating a correction signal from the region of correction; and (d) using the correction signal to correct a measured signal from one or more regions of interest. This invention also provides a method for defining a region of correction for use in a method for correcting the signal in an image having a plurality of regions of interest, the defining method comprising the steps of: (a) providing an image having a plurality of regions of interest; (b) extracting geometric information for a plurality of regions of interest; (c) selecting a location between at least two regions of interest; (d) selecting at least one parameter to describe regions of correction; and (e) constructing regions of correction between the at least two regions of interest.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to a method for correcting signals detected by adetection system in a diagnostic instrument.

2. Discussion of the Art

Raw images generated by a diagnostic instrument having a digital imagesensor as a detector, such as, for example, the Applied Biosystems Prism7000 diagnostic instrument, can exhibit an anomaly known as“cross-talk.” Cross-talk refers to the situation where a signal from agiven location in the image (for example, a given well in a plate havinga plurality of wells, e.g., a 96-well PCR plate), causes a variation inthe signal at a different location in the image (for example, adifferent well in the plate having a plurality of wells). A specificregion within an image associated with an independent signal is oftenreferred to as a region of interest (alternatively referred to herein asROI). Each ROI defines the specific pixels within the image associatedwith a specific reaction. Variations in signal due to cross-talk,although typically small, can induce variations in reactionquantification of one or more regions of interest within the image. Insome cases, sensitivity of the reaction is reduced by requiring anincrease in signal threshold in order to avoid false positive resultsdue to cross-talk.

The areas in the image between the regions of interest of the imagecontain optical information that can be used to compensate for sourcesof variation in signal. These sources of signal variation can resultfrom a specific geometric optical reflection, scattered light fromoptical components, light leakage, changes in intensity of the source oflight, and the like. All of these sources of variation can contribute toa dynamically changing error in the optical signal in a given region ofinterest of the image.

It is desired to monitor a region of interest associated with a reactionand ultimately correct for varying anomalous signals over the course ofa testing run in a diagnostic instrument.

SUMMARY OF THE INVENTION

In one aspect, this invention provides a method for correcting thesignal in an image having a plurality of regions of interest, the methodcomprising the steps of:

-   -   (a) providing an image having a plurality of regions of        interest, these regions of interest having areas between them;    -   (b) determining a region of correction between at least two        regions of interest;    -   (c) calculating a correction signal from the region of        correction; and    -   (d) using the correction signal to correct a measured signal        from one or more regions of interest.

In another aspect, this invention provides a method for defining aregion of correction for use in a method for correcting the signal in animage having a plurality of regions of interest, the defining methodcomprising the steps of:

-   -   (a) providing an image having a plurality of regions of        interest;    -   (b) extracting geometric information for a plurality of regions        of interest;    -   (c) selecting a location between at least two regions of        interest;    -   (d) selecting at least one parameter to describe regions of        correction; and    -   (e) constructing regions of correction between the at least two        regions of interest.        The regions of correction defined in the forgoing method can be        stored for further use to correct signals measured from one or        more regions of interest.

The specified regions of correction can have various shapes, such as,for example, circles, squares, diamonds, rectangles, or other geometricfigures. Storing of the regions of correction involves determining thedefinition of the location and the shape of the geometric areas andspecifying the pixels contained within each area.

The method of this invention can be used to measure a dynamicallychanging signal and the effect of the dynamically changing signal on aregion of interest of a specific reaction. Correcting for the cross-talkinherent in a dynamically changing signal will greatly increase thesensitivity of the method of detection used in an assay employing suchsignals. The method of this invention does not affect the optical pathof the light collected by a detector. The method can be applied directlyto an image that is collected for all the regions of interest

By measuring the signals in the regions of correction, the signalanomaly due to cross-talk can be significantly reduced.

The sizes and shapes of the regions of correction can vary and dependprimarily on the orientation of the existing regions of interest and anyimage distortions that may be present.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a flow chart that defines the placement of diamond-shapedregions of correction about circular reaction regions of interest.

FIG. 2A illustrates one embodiment of reaction regions of interest andregions of correction. The reaction regions of interest are circular andthe regions of correction are circular. FIG. 2B illustrates anotherembodiment of reaction regions of interest and regions of correction.The reaction regions of interest are circular and the correction regionsof interest are rectangular. FIG. 2C illustrates still anotherembodiment of reaction regions of interest and regions of correction.The reaction regions of interest are circular and the regions ofcorrection are shaped like diamonds.

FIG. 3 is a flow chart illustrating the application of the image-basedcorrection algorithm of this invention.

FIG. 4 is a map of a 96-well plate illustrating the location of eachpositive response and each negative response.

FIG. 5 is a sample of an image at the end of a run.

FIG. 6 is a sample of an image showing reaction regions of interest andregions of correction.

FIG. 7 shows fluorescence responses in a PCR assay without image-basedcorrection applied.

FIG. 8 shows fluorescence responses in a PCR assay without image-basedcorrection applied. In this figure, the Y scale is expanded.

FIG. 9 shows fluorescence responses in a PCR assay with image-basedcorrection. In this figure, the Y scale is expanded.

FIG. 10 shows the fluorescence response in a PCR assay for well F-11from FIG. 4 with and without assay-based correction.

DETAILED DESCRIPTION

As used herein, the expression “region of interest” means the collectionof pixels within an image that define the location of a specific opticalsignal. The expression “reaction region of interest” means the region ofinterest associated with a specific reaction in an assay. Theexpressions “region of correction” and “correction region of interest”mean the area associated with the background portion of the imageadjacent to a reaction region of interest. The expression “reactionpixel sum” means the sum of all the pixel intensity values within areaction region of interest. The expression “reaction pixel count” meansthe number of pixels within a reaction region of interest. Theexpression “reaction region of interest pixel average” means the valueobtained by dividing the reaction pixel sum by the reaction pixel count.The expression “correction pixel sum” means the sum of all the pixelintensity values within a region of correction. The expression“correction pixel count” means the number of pixels with a region ofcorrection. The expression “region of correction pixel average” meansthe value obtained by dividing the correction pixel sum by thecorrection pixel count. The term “scale” means a multiplicative factorapplied to the correction calculation. The term “centroid” means thegeometric center of a region of interest. As used herein, the terms“circular”, “rectangular”, “annular”, and other terms relating to shapereferred to herein are intended to include shapes that are substantiallycircular, substantially rectangular, substantially annular, and shapesthat are substantially similar to the other shapes referred to herein,respectively.

In one aspect, this invention provides a method for correcting an imagehaving a plurality of reaction regions of interest and a plurality ofregions of correction, the method involving the steps of:

-   -   (a) providing an image having a plurality of regions of        interest, these regions of interest having areas between them;    -   (b) determining a region of correction between at least two        regions of interest;    -   (c) calculating a correction signal from the region of        correction; and    -   (d) using the correction signal to correct a signal measurement        from one or more regions of interest.        Prior to carrying out the method of this invention, certain        steps must be taken to calibrate the imaging system, which is        typically a digital imaging system. FIG. 3 shows a flow chart        that illustrates steps for defining regions of correction        between adjacent reaction regions of interest for the        calibration step of the method of this invention. In this flow        chart, generic regions of correction are described.

According to the calibration method of this invention, the centroid ofeach reaction region of interest is determined. The reaction regions ofinterest are typically determined by using a calibration where signalsin a device having a plurality of reaction sites are measured. A signalis measured at each reaction site. In the case of 96-well reactionplates, the signals in a calibration plate containing fluorescent dye ateach reaction site can be measured by an imaging sensor. A calibrationplate is a 96-well reaction plate used for calibrating the instrumentused. The reaction regions of interest can be determined by locating thecontiguous pixels at each reaction site within the image. The geometriccentroid of each set of centroids from four adjacent reaction regions ofinterest can be used to determine a center point for a region ofcorrection. A region of correction using that center point and aspecific geometric shape can be defined. As shown in FIGS. 2A, 2B, and2C, the reaction regions of interest are circular in shape. A region ofcorrection can be circular-shaped, as shown in FIG. 2A,rectangular-shaped, as shown in FIG. 2B, or diamond-shaped, as shown inFIG. 2C. Other shapes, such as, for example, closed polygons, aresuitable for both the reaction regions of interest and the regions ofcorrection. The parameters of the regions of correction are typicallyradii of rings for circular-shaped regions of correction, length andwidth for rectangular-shaped regions of correction, and length of sidesfor diamond-shaped regions of correction. Dimensions for the particulargeometric shape selected are specified. An alternative to definingregions of correction by means of geometric shapes involves the use ofan arbitrary bitmap. Such a bitmap could, for example, be a 9 by 9 arrayof values specifying which pixels would be included in the region ofcorrection and which pixels would be excluded from the region ofcorrection. The center points of the regions of correction can bemirrored to create regions of correction on the periphery of the platefor the outer rows and columns of the reaction regions of interest inthe image. Thus, in the case of diamond-shaped regions of correction inan image having 96 reaction regions of interest, there are 117diamond-shaped regions of correction of interest in total, i.e., four(4) diamond-shaped regions of correction per reaction region ofinterest. The use of diamond-shaped regions of correction is shown inFIG. 2C. The regions of interest associated with specific wells can bedetermined and stored, such as, for example, by means of a computer. Inthis embodiment, each reaction region of interest has the four adjacentregions of correction associated with it.

Similarly, in the case of rectangular-shaped regions of correction in animage having a plurality of reaction regions of interest (e.g., 96 wellsin a plate), the rectangles can be oriented with the length parallel tothe x-axis or to the y-axis, as shown in FIG. 2B. For the x-direction(horizontal), the center point between two adjacent regions of interestis located. A rectangle is constructed by using the center point betweentwo adjacent regions of interest as the center of the region ofcorrection between the regions of interest. For the y-direction(vertical), the center point between two adjacent regions of interest islocated. The rectangle is constructed by using the center point betweentwo adjacent regions of interest as the center of the region ofcorrection between the regions of interest. Rectangles are also createdon the periphery of the image for the outer rows of regions of interestand outer columns of regions of interest. The mirror of the centerbetween adjacent regions of interest is used to set the center of theregion of correction rectangle. The regions of interest associated withspecific wells can be determined and stored. In this embodiment, eachreaction region of interest has the four adjacent regions of correctionassociated with it. Measures other than the centroid of the regions ofcorrection can also be used to define the location of regions ofcorrection. For example, the region of correction can be placedequidistant from boundaries of adjacent regions of interest.

After the region of correction calibration is performed, the correctionbased upon from the region of correction can be applied by using thefollowing method. Once the region of correction calibration isperformed, a background offset value needs to be generated. This valuecan be generated in at least two ways. According to a first alternative,a background calibration can be performed. In this method, an image istaken of a plate without any fluorescent dye. During the backgroundcalibration, the average pixel value for each region of correction iscalculated by dividing the pixel sum by the pixel count in that regionof correction to obtain an average pixel value. This average pixel valueis indicative of the background light level and is referred to as thebackground offset value. The background offset values are stored for usein future runs, e.g., PCR runs. Alternatively, the background offsetvalue can be determined on a run-by-run basis by calculating the averagepixel value for each region of correction for the first reading of arun, e.g., a PCR run. Because the first (or first few) readings of a PCRrun occur before a significant reaction signal is produced, thisalternative method provides a good representation of the background.

The signal correction is performed in the following manner. Performanceof signal correction is depicted in FIGS. 1 and 3. For each reaction,the reaction pixel sum and the reaction pixel count are calculated byusing the reaction region of interest. The average pixel value for thefour regions of correction associated with a given reaction region ofinterest is calculated. Although four regions of correction are shown inFIG. 1 and diamond-shaped regions of correction are shown in FIG. 1, themethod is not limited to four regions of correction nor is the inventionlimited to diamond-shaped regions of correction. The background offsetvalue is subtracted from the region of correction pixel average. Then,this difference is multiplied by the reaction region of interest pixelcount and, if necessary, by a scale factor, to generate a correctionvalue. The correction value is then subtracted from the reaction regionof interest pixel sum to generate a corrected reaction region ofinterest pixel sum. The scale factor is typically dependent upon thedetection system. An example of a scale factor is 1.15. In someinstrument systems, multiple exposures are made at each reading toincrease the dynamic range of measurement. In this case, a correspondingbackground offset and region of correction pixel average needs to begenerated for each exposure. The correction to the reaction pixel sum isthen made for the exposure of longest duration that does not exhibitsignificant saturation of the image sensor.

This invention can also be applied to an assay system based on array ora microarray, such as, for example, the Vysis GenoSensor genomic DNAmicroarray system (Abbott Laboratories, Abbott Park, Ill.). Such systemscan measure a plurality of genomic targets through hybridization to anarray of capture targets placed on a surface, such as, for example, aglass “chip” or a microscope slide. The product of the hybridization istypically measured by means of fluorescent dyes and an electronicimaging system.

The following non-limiting example further explains the method of thisinvention.

EXAMPLE

A real time PCR run for HIV was performed on an ABI Prism 7500instrument (Applied Biosystems, Foster City, Calif.). This instrumentutilizes a 96-well plate format with wells arranged in a 12×8 array. Therun was configured so that there were 84 wells containing positivesamples with a concentration of 1×10⁶ copies/mL and 12 wells notcontaining positive samples, i.e., negative wells. The negative wellswere distributed on the plate to maximize the potential cross-talk fromthe wells containing positive samples. FIG. 4 illustrates the layout ofthe plate.

The ABI Prism 7500 instrument uses a CCD camera and measuresfluorescence in five wavelength bands. FIG. 5 shows one image from theend of the PCR run. FIG. 6 shows the same image with the reactionregions of interest and the regions of correction superimposed. In thisexample, a diagonal array of diamond-shaped regions of correction, eachof which contained of 25 pixels, were used. The first reading in the PCRrun was used to establish the background offset values for eachsubsequent reading. The scaling factor used was 1.15.

FIG. 7 shows the raw fluorescence signals for all 96 samples without anyimage-based correction applied. As can be seen, the 84 positive samplesgenerated signals significantly above the background fluorescence bycycle 15 and approached their maximum fluorescence by cycle 25. FIG. 8shows the same responses with the Y-axis scaled to focus on theresponses in wells not containing positive samples. All of the negativeresponses showed a small but significant rise from cycles 15 through 25,which is caused by cross-talk from the responses of the positivesamples. FIG. 9 shows the effect of the image-based correction on thenegative responses. As can be seen, the cross-talk signal has beeneffectively eliminated. FIG. 10 shows the response for well F-11 withand without the image-based correction applied.

The method is also applicable to images that contain a fewer or agreater number of regions of interest.

Various modifications and alterations of this invention will becomeapparent to those skilled in the art without departing from the scopeand spirit of this invention, and it should be understood that thisinvention is not to be unduly limited to the illustrative embodimentsset forth herein.

1. A method for correcting the signal in an image having a plurality ofregions of interest, the method comprising the steps of: (a) providingan image having a plurality of regions of interest, these regions ofinterest having areas between them; (b) determining a region ofcorrection between at least two regions of interest; (c) calculating acorrection signal from the region of correction; and (d) using thecorrection signal to correct a signal measurement from one or moreregions of interest.
 2. The method of claim 1, further including thestep of determining a background signal and adjusting the correctionsignal of a run by subtracting the background signal from the correctionsignal.
 3. The method of claim 2, wherein the background signal is abackground signal stored prior to commencing the run.
 4. The method ofclaim 2, wherein the background signal is a background signal determinedduring the run.
 5. The method of claim 1, wherein the correction signalis scaled.
 6. The method of claim 1, wherein the regions of correctionhave a plurality of sides.
 7. The method of claim 1, wherein the regionsof correction have four sides.
 8. The method of claim 1, wherein theregions of correction are closed polygons.
 9. The method of claim 1,wherein the regions of correction are circular.
 10. The method of claim1, wherein the regions of correction are annular.
 11. The method ofclaim 1, wherein the regions of correction are defined by a bitmap. 12.The method of claim 1, wherein said plurality of regions of interest arefrom a multi-well plate.
 13. The method of claim 1, wherein athermocycler reader is employed.
 14. The method of claim 1, furtherincluding the step of storing the regions of correction defined in step(c).
 15. A method for defining a region of correction for use in amethod for correcting the signal in an image having a plurality ofregions of interest, the defining method comprising the steps of: (a)providing an image having a plurality of regions of interest; (b)extracting geometric information for the plurality of regions ofinterest; (c) selecting a location between at least two regions ofinterest; (d) selecting at least one parameter to describe regions ofcorrection; and (e) constructing regions of correction between the atleast two regions of interest.
 16. The method of claim 15, furtherincluding the step of constructing additional regions of correction thatare not between the at least two regions of interest.
 17. The method ofclaim 16, wherein a sufficient number of additional regions ofcorrection are constructed so that each region of interest has the samenumber of regions of correction as does any other region of interest.18. The method of claim 15, wherein the geometric information for theregions of interest is a centroid.
 19. The method of claim 18, whereinthe location between at least two regions of interest is the centerpoint between the centroids of at least two regions of interest.
 20. Themethod of claim 15, wherein the regions of correction are selected fromthe group consisting of polygons, circles, annuli, and bitmaps.
 21. Themethod of claim 15, further including the step of storing the regions ofcorrection associated with the at least two regions of interest.